A. Non-Phosphodiester Polynucleotide Analogs
DNA consists of covalently linked units, each composed of a nucleobase (adenine, cytosine, guanine, or thymine) attached to a pentose sugar (deoxyribose) via a glycosidic linkage, with a phosphate ester (phosphodiester) linking successive sugar rings. Numerous types of DNA analogs have been synthesized, with most variations typically having a modification or replacement of the phosphodiester backbone.
Examples of polynucleotide analogs having a modified phosphate backbone include: methylphosphonates, phosphorothioates, phosphoramidites, phosphorodithioates, phosphorotriesters, and boranophosphates. An alternative approach is the development of structural mimetics of the phosphodiester linkage, generally with the objective of providing a backbone linkage that is charge neutral (to increase the stability of DNA hybrid complexes), relatively hydrophobic (to increase cellular uptake), and achiral. Examples of polynucleotide analogs wherein the phosphodiester backbone is replaced by a structural mimic linkage include: alkanes, ethers, thioethers, amines, ketones, formacetals, thioformacetals, amides, carbamates, ureas, hydroxylamines, sulfamates, sulfamides, sulfones, glycinylamides, and others.
In addition to replacing the phosphodiester linkage, alternative approaches have replaced the entire (deoxy)ribose-phosphate backbone, retaining just the nucleobases. One of these approaches replaces the entire (deoxy)ribose-phosphate backbone with a peptide-like backbone, generating a so-called "peptide nucleic acid," "polyamide nucleic acid," or simply "PNA" (Nielsen et al. (1991) Science 254: 1497; Nielsen et al. (1994) Bioconj. Chem. 5: 3; Leijon et al. (1994) Biochemistry 33: 9820; Huang et al. (1991) J. Org. Chem. 56: 6007; Egholm et al. (1993) Nature 365: 556; Buchardt et al. (1993) Trends Biotechnol. 11; 384; Nielsen P E (1995) Rev Biophys Biomol Struct 24: 167; Agrawal et al. (1995) Curr Opin Biotechnol. 6: 12; Nielsen et al. (1993) Anticancer Drug Res. 8: 53; Cook P D (1991) Anticancer Drug Des. 6: 585). PNAs have an achiral, noncharged backbone, as exemplified by a backbone composed of N-(2-aminoethyl)glycine units, which is a suitable structural mimic of DNA. Hybrids between such a PNA and complementary sequence DNA or RNA are reported to exhibit higher thermal stability per base pair than DNA:DNA or RNA:RNA duplexes (Wittung et al. Nature 368: 561).
PNAs have been reported to have many interesting properties. Binding of PNA to double-stranded DNA occurs by strand invasion via formation of a D-loop strand displacement complexes (Egholm et al. (1993) Nature 365: 556) that have unique biological properties, including the capacity to serve as artificial transcription promoters in some contexts (Mollegaard et al. (1994) Proc. Natl. Acad. Sci. (U.S.A.) 91: 3892). PNAs have been shown to bind to DNA and RNA in a sequence-dependent manner (Brown et al. (1994) Science 265: 777; Egholm et al. (1993) op.cit), and exhibit superior base pair mismatch discrimination in PNA/DNA hybrids than do DNA/DNA duplexes (Orum et al. (1993) Nucleic Acids Res 21: 5332).
PNAs have been used to target the single strand-specific nuclease S1 to a PNA/DNA hybrid formed via strand invasion, making S1 nuclease act like a pseudo restriction enzyme (Demidov et al. (1993) Nucleic Acids Res 21: 2103). Alternatively, complementary PNAs have been used to block sequence-specific DNA restriction enzyme cleavage of dsDNA plasmids (Nielsen et al. (1993) Nucleic Acids Res 21: 197). PNAs have been used to arrest transcription elongation by targeting a complementary sequence PNA to the template DNA strand (Nielsen et al. (1994) Gene 149: 139). PNA strand invasion has also been shown to inhibit transcriptional activation by the transcription factor NF-.kappa.B by blocking its interaction with 5' regulatory sequences to which it normally binds (Vickers et al. (1995) Nucleic Acids Res 23: 3003). Interaction of certain DNA-binding ligands with PNA/DNA hybrids has also been reported (Wittung et al. (1994) Nucleic Acids Res 22: 5371).
The antisense and antigene properties of PNAs have been reported (Bonham et al. (1995) Nucleic Acids Res 23: 1197; Hanvey et al. (1992) Science 258: 1481; Nielsen et al. (1993) AntiCancer Drug Des 8: 53). A vector-mediated delivery method for introducing PNAs through phospholipid membranes and through the blood-brain barrier have been reported (Pardridge et al. (1995) Proc. Natl. Acad. Sci. (U.S.A.) 92: 5592; Wittung et al. (1995) FEBS Lett 375: 27). Orum et al. (1995) Biotechniques 19: 472 report a method for sequence-specific purification of nucleic acids by PNA-controlled hybrid selection.
B. Telomerase and Telomerase-Related Proteins
The DNA at the ends of telomeres of the chromosomes of eukaryotes usually consists of tandemly repeated simple sequences. Telomerase is a ribonucleoprotein enzyme that synthesizes one strand of the telomeric DNA using as a template an 11 nucleotide sequence contained within the RNA component of the enzyme (see Blackburn (1992) Annu. Rev. Biochem. 61:113-129). The RNA component of human telomerase has been cloned and sequenced (Feng et al. (1995) Science 269: 1267 and U.S. Pat. No. 5,583,016).
Despite the seemingly simple nature of the repeat units of telomeric DNA, scientists have long known that telomeres have an important biological role in maintaining chromosome structure and function. More recently, evidence consistent with a loss of telomeric DNA acting as a trigger of cellular senescence and aging indicates that regulation of telomerase may have important biological implications. (see Harley (1991) Mutation Research 256: 271-282). In addition, telomerase activity is detected in 85-95% of human tumors, and is required for sustained tumor proliferation. Maintenance of telomere length in tumor cell lines can be prevented by the expression of antisense RNA complementary to the hTR, leading to cell crisis (Feng et al. (1995) Science 269: 1236), and telomerase inhibition is believed to suppress tumor growth.
Methods for detecting telomerase activity, as well as for identifying compounds that regulate or affect telomerase activity, together with methods for diagnosing of cellular senescence and immortalization by controlling telomere length and telomerase activity, have been described. See International Patent Application Publication Nos. WO 95/13381; WO 95/13382; and WO 93/23572. Polynucleotide primers and probes that specifically hybridize to the RNA component of mammalian telomerase are also described in U.S. Pat. No. 5,583,016; U.S. patent application Ser. No: 08/272,102, filed Jul. 7, 1994; Ser. No. 08/472,802, filed Jun. 7, 1995; Ser. No. 08/482,115, filed Jun. 7, 1995; Ser. No. 08/521,634, filed Aug. 31, 1995; Ser. No. 08/630,019, filed Apr. 9, 1996; Ser. No. 08/660,678, filed Jun. 5, 1996; and International Patent Application Publication No. WO 96/01835. Such polynucleotides can be used to detect the RNA component of telomerase and inhibit telomerase activity, particularly in cancer cells. For example, DNA oligonucleotides can inhibit telomerase, but this inhibition requires that oligonucleotides be relatively long (18-40 nucleotides) and be present at relatively high concentrations (Collins et al. (1995) Cell 81: 677; Shippen-Lentz and Blackburn (1990) Science 247: 546).